Modest elevations in plasma homocysteine are associated with an increased risk of atherosclerotic disease. Impaired endothelial vasodilator function appears to play an important role in progression and complications of atherosclerosis. Thus, homocysteine-induced endothelial dysfunction is a plausible mechanism for predisposition to atherosclerosis. In previous studies funded by this R01, we have demonstrated that induction of experimental hyperhomocysteinemia by methionine loading rapidly produces profound endothelial dysfunction in human subjects, which can be reversed by anti-oxidants. We have also shown that high protein diets cause post-prandial hyperhomocysteinemia. In addition, we have developed a series of novel pharmacological models to characterize endothelial mediators and several putative mechanisms of oxidant stress. We therefore plan to test the following specific aims: 1) To explore endothelial mediators affected by experimental hyperhomocysteinemia in humans (specifically test the hypothesis that homocysteine selectively impairs nitric oxide activity and that there is compensatory upregulation of endothelium-derived hyperpolarizing factor); 2) To test potential oxidant mechanisms underlying the endothelial dysfunction (specifically xanthine oxidase, cyclooxygenase and inducible nitric oxide synthase); and 3) To examine the effect of dietary interventions with varying protein and folate intakes on plasma homocysteine and endothelial function, particularly in subjects with genetic (MTHFR mutation) or renal (transplant recipients) predisposition to hyperhomocysteinemia. Endothelial function will be tested in conduit and resistance vessels in vivo using flow mediated vasodilatation and forearm blood flow responses to intra-arterial administration of vasoactive agents. This patient-orientated research should lead to new understanding of the pathophysiological mechanisms linking homocysteine to vascular disease in humans. The results may also suggest novel potential therapeutic approaches to be tested in future outcome trials.